Over past decades, the prior art has offered several types of rapid diagnostic testing techniques primarily for body fluids such as whole blood, serum, plasma, urine, spinal fluid, amniotic fluid, mucous, saliva, and the like for the presence of infection or other conditions such as pregnancy, abused drugs and cardiovascular disorders such as acute myocardial infarction (AMI).
The first of these tests were the Latex Particle Agglutination tests, then the Flow Through tests leading to the current Lateral Flow Single Step test. Such tests typically utilize well known sandwich-type immunoassay techniques. In such tests a fluid specimen is supplied from a subject which could carry at least one analyte, such as an antibody, which is specific to the condition being tested. The fluid specimen is exposed to a conjugate having at least one mobilizable binding member, such as an antigen (or antibody in some cases), which has immuno-determinant(s) (or specific binding sites for the immuno-determinant(s) in certain cases) of the analyte in question. The binding member is conjugated to a visibly detectable label such as colloidal gold. During exposure of the specimen to the conjugate, an immuno-chemical reaction occurs wherein the analyte in question binds to the binding member to form the first affinity binding labeled analyte complex.
The fluid containing the labeled analyte complex is then directed to flow into a reaction membrane having a zone coated with at least one immobilized capture binding member which is similarly immuno-determinant of the analyte in question. As the fluid passes through the membrane, a second immuno-chemical reaction occurs wherein the labeled analyte complex binds to the capture binding member to form the second affinity binding immuno-sandwich complex. The accumulation of the secondly bound immuno-sandwich complex beyond a threshold amount in the zone creates a colored test line, the so-called “T-line”. The reaction membrane typically has a second zone located further downstream from the T-line as an internal system control line, the so-called “C-line”. The control line is used as an internal indicator of functional validity.
Unfortunately, depending on the type of condition being detected, these tests provide a typical accuracy of between 85% and 99%, falling short of the 99.5% or above accuracy generally considered to be necessary for a confirmatory test. The reasons for the insufficient accuracy are primarily due to the lack of overall higher sensitivity and specificity of the apparatus. Different samples may contain chemicals or particles which interfere with or inhibit the fluid flow or otherwise interfere with one or both of the affinity binding reactions. Prior apparatuses have attempted to enhance sensitivity or specificity by pretreating various parts of the apparatus with reaction or flow enhancing reagents, pH conditioning chemicals, or even non-specific adhesive blocking molecules which will “block-out” non-analyte molecules which might cause non-specific adhesion, or otherwise compete with the analyte in question for specific binding members, especially on the reaction membrane. These attempts have met with limited success in some types of testing, but do not provide the desired accuracy in many others. Also, pretreatment with two or more of the above pretreatments exacerbates the difficulties in obtaining uniform manufacturing due to potential incompatibilities between the pretreatment chemicals. For example, the pH conditioner might disrupt the effectiveness of the non-specific blocking member molecules. Or, the manufacturing step of pretreating with the second pretreatment chemical can dislodge some of the first pretreatment chemical.
Further, lot-to-lot variation in the manufacture of the test apparatuses can often lead to ambiguous results, such as false negatives as well as weak false positives, so-called “ghost lines” or “phantom lines”. False negatives typically occur when non-specific molecules interfere with the first and/or second affinity binding actions. It has been found that non-analyte molecules can clump together in fluid samples that are not well mixed so that they temporarily prevent access between analytes and binding members. Even temporary interference can prevent an adequate number of labeled analyte complexes and/or ultimately immuno-sandwich complexes from forming. In this way, if a non-analyte molecule or clump of molecules blocks access between analytes and binding members for only a few seconds, it may be enough to induce a false negative result. Further, clumps of non-analyte molecules can carry an overabundance of the labeled mobilizable binding members to the second affinity binding site to generate a false positive.
Chemically non-uniform flows can result in flows having non-uniform first affinity binding by the time they reach the reaction membrane leading to inaccuracies. Such non-uniform flows can be caused by a number of factors. First, some portions of the fluid may flow faster than others from time to time. In those tests having deposits of dried reagent, faster flows tend to reach the dried reagent first. These flows tend to exhibit a greater degree of first affinity binding per unit fluid or at least uptake of mobilizable labeled binding members, and can potentially carry a greater concentration of clumps of non-analyte molecules which can carry away labeled mobilized binding members. Further, the deposit of dried reagent itself can exhibit portions of higher concentration than others resulting in similar chemical nonuniformity in the flow. Other flows having a lower than average concentration of analyte molecules, and/or having a greater concentration of non-clumped, non-analyte molecules which merely inhibit analyte binding but do not carry away mobilizable labeled binding members, exhibit less apparent first affinity binding. These flow and concentration dis-uniformities are responsible for many of the unsatisfactory results discussed above.
Therefore, although these prior devices provide a convenient, quick, economic, and simplified way to conduct such testing without requiring sophisticated instrumentation or trained professionals, in many settings these rapid tests are useful only for preliminary screening purposes, not as a confirmatory test. To this day, for example, the Western Blot Analytical Assay is the only one reliably used for the confirmatory detection of HIV infection in a clinical laboratory setting worldwide. Due to its multi-step manipulation and verification phases, completion of this type of assay takes days, if not weeks. Such a delay can unfortunately lead to further propagation of infectious pathogens such as HIV or other serious results, such as the metastasis of cancers. There is virtually no generally accepted practical or economical confirmatory rapid diagnostic testing technique for use in a point-of-care setting to rapidly detect serious diseases such as HIV infection and AMI, available in the market place today.
Therefore, there is a need to refine the accuracy and expedite the performance of prior immunoassay chromatographic rapid testing apparatuses to a higher and new level for use in the speedy and early detection and confirmation of the presence of pathogens or pathogenic conditions such as occurs with HIV infection, cancers and other disorders.